Vitamin B.sub.12 (cyanocobalamin) has an empirical formula of C.sub.63 H.sub.88 O.sub.14 N.sub.14 PCo (Mol. Wt. 1355.4 daltons) and is a complex molecule having three major components: a porphyrin-like ring structure consisting of four reduced and extensively substituted pyrrole rings (designated A to D) surrounding a single cobalt atom; an .alpha.-5,6-dimethylbenzimidazole ribonucleotide group which links to the corrin nucleus with bonds to the cobalt atom and to the propionate side chain of the D ring; and a variable R group which can be cyanide, hydroxyl, methyl or 5-deoxyadenosyl. The cyano-substitution is an artifact of isolation but is the form which is currently assayed. Hydroxycobalamin, methylcobalamin and 5-deoxyadenosylcobalamin are the physiologically active coenzyme forms of vitamin B.sub.12. The two major active cobalamins, methylcobalamin and 5-deoxyadenosylcobalamin, are essential for DNA biosynthesis, replication and cell growth.
Dietary vitamin B.sub.12, in the presence of gastric acids and pancreatic proteases, is released from intestinal R protein-like binding proteins and is immediately bound to intrinsic factor (IF), the gastrointestinal binding glycoprotein responsible for the specific binding and absorption of vitamin B.sub.12. Intrinsic factor has a molecular weight of approximately 40,000 and differs markedly in structure from the R proteins. Its ability to bind vitamin B.sub.12 at the exclusion of metabolically inactive cobinamide has led to the predominant use of this binding protein in the commercially available vitamin B.sub.12 assays. The IF:vitamin B.sub.12 complex interacts with the ileal mucosal cells and is then absorbed into the blood stream.
Following absorption, all three active cobalamins are present in plasma bound to plasma-binding proteins known as transcobalamin II and R proteins (also known as nonintrinsic factors). lranscobalamin II binds approximately 20% of the total plasma vitamin B.sub.12 while the remainder is bound to the R proteins. Vitamin B.sub.12 bound to transcobalamin II, is rapidly cleared from the plasma and delivered to a variety of cells, such as the hepatic parenchymal cells of the liver. The R proteins may also play a role in the delayed transport and cellular delivery of vitamin B.sub.12 to the liver. Because of the high affinity with which the plasma-binding proteins bind vitamin B.sub.12, the denaturation of these proteins is necessary in the performance of cyanocobalamin assays.
Vitamin B.sub.12 deficiency is due primarily to a disruption of IF:vitamin B.sub.12 binding resulting from a failure of parietal cell production of intrinsic factor or the production of anti-intrinsic factor antibodies. The failure of parietal cells is probably due to the presence of cytotoxic autoantibodies. Serum antibodies to intrinsic factor have been extensively studied. Two types of antibodies have been identified: the blocking antibody (Type I), which blocks the binding of vitamin B.sub.12 to IF and a nonblocking antibody (Type II), which reacts with the IF:vitamin B.sub.12 complex. Serum blocking antibodies are present in 50-76% of vitamin deficient patients and have a high degree of specificity for the diagnosis of pernicious anemia. Commercial assay kits which measure the levels of these anti-IF antibodies are available (Corning ImmoPhase IFAB) and studies on the utility of these assays in the differential diagnosis of vitamin B.sub.12 deficiency have proceeded to the present.
Vitamin B.sub.12 deficiency is more commonly caused by defective gastrointestinal absorption than from a deficient diet. Therefore, additional factors which can lead to vitamin B.sub.12 malabsorption include the combination of gastric achlorhydria and decreased secretion of IF secondary to gastric atrophy or surgery, intestinal parasites, bacterial gastroenteritis and intestinal malignancies. Other disorders which may account for a vitamin B.sub.12 deficiency include dietary deprivation (veganism), drugs, organ disease (liver), hyperthyroidism and transcobalamin II aberrations.
A deficiency in vitamin B.sub.12 can lead to disease states marked by defective DNA synthesis culminating in delayed cellular mitosis. Since the cells having the most rapid metabolism will be the most dramatically affected, the hematopoietic system is particularly sensitive to vitamin B.sub.12 deficiencies. The primary clinical manifestion of this vitamin deficiency is the development of a megaloblastic anemia which is also known as pernicious anemia. Vitamin B.sub.12 is also implicated in the maintenance of the myelin of the nervous system. For these reasons, it is very important to be able to diagnose vitamin B.sub.12 deficiencies.
The current state of the art regarding vitamin B.sub.12 assays utilizes a wide variety of assay techniques. Microbial assays are based upon the principle that some microorganisms such as Euglena gracilis and Lactobacillus leichmannii require vitamin B.sub.12 for normal growth and replication. Standard curves are produced using known amounts of vitamin B.sub.12 and bacterial growth produced by a sample containing an unknown amount of vitamin B.sub.12 is compared with the standard curve. Bacterial growth can be measured in numerous ways but the most common is turbidimetric measurement.
The deoxyuridine suppression test is performed on bone marrow cells which have been aspirated from the patient. A vitamin B.sub.12 deficiency is noted if there is a reduction in the suppression of radioactive iododeoxyuridine incorporation into DNA by deoxyuridine.
High-performance liquid chromatography is based upon the variability in the rate of travel of different compounds through a medium under pressure within a column. The elutes are then detected at picogram/ml concentrations by ultra-violet or other detectors such as fluorescence and electrochemical detection.
Another method involves the use of antisera specific to vitamin B.sub.12 which are employed in specific radioimmunoassays to directly measure the liberated vitamin.
Still another method involves competitive protein-binding radioimmunoassays (radioisotopic dilution assays). These assays have the prerequisite that the vitamin B.sub.12 in the sample be freed from its binding proteins by physical, chemical or enzymatic denaturation or digestion. The most commonly used methods are the boil method which involves boiling the sample for 15-30 minutes and the non-boil method which involves the use of highly alkaline buffer solutions containing KCN which simultaneously denature the binding proteins and convert the cobalamins to the stable cyanocobalamin form. Both the boil and the non-boil methods use radioactive (.sup.57 Co-labelled) vitamin B.sub.12 in a competitive system which employs purified hog intrinsic factor as the binding agent and a separation step to remove unbound sample vitamin B.sub.12.
In spite of the fact that there are numerous vitamin B.sub.12 assays currently available, there remains a continuing need for an improved assay that will overcome the shortcomings of the state of the art techniques. These shortcomings include a lack of sensitivity, difficulties encountered in standardization and the impracticality of use in high volume situations, i.e. in hospitals. Further, there are no commercial non-isotopic immunoassays for this analyte on the market. During the past decade, major attempts have been made by numerous groups to produce such a non-isotopic test for vitamin B.sub.12. These attempts have been unsuccessful, due primarily to the small size and complexity of the vitamin B.sub.12 molecule. Therefore, there is a current need for an accurate, precise and reliable reference method for assaying cyanocobalamin. This invention is intended to address that need.